Polymyxins cause the death of bacteria by disrupting the cell membrane of many Gram-negative bacilli [1]. After being discovered in the late 1940s, polymyxins were used for treatment for many years, but their use was discontinued in the 1970s due to their side effects [2]. Since the 1970s and 1980s, more effective antibiotics with fewer side effects have been used for treatment. For a long time, carbapenem antibiotics have been the most important drug in the treatment of multidrug-resistant Gram-negative bacilli (MDRGNB). In recent years, polymyxin group antibiotics have been included in the treatment protocol again, due to the development of resistance to the carbapenem group of antibiotics. With the reintroduction of polymyxin group antibiotics into the treatment protocol, an increasing resistance has developed to this group of antibiotics.
For colistin susceptibility tests, broth microdilution (BMD), broth disk elution, and colistin agar tests are acceptable by the Clinical and Laboratory Standards Institute (CLSI), but for polymyxin B, BMD is the only approved method. Other testing methods, such as disk diffusion and gradient diffusion, are not currently recommended [3]. The current method accepted by both the CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [4] is the reference BMD method. In the polymyxin group, only polymyxin B and colistin are in clinical use [5]. In the minimal inhibitory concentration (MIC) measurements, the use of colistin sulfate and polymyxin sulfate is recommended. The CLSI [3] has published the clinical breakpoints for colistin and polymyxin B against intermediate and resistant
In our study, we aimed to investigate the MIC value of both colistin and polymyxin B against MDRGNB strains; ESBL positive
A total of 268 MDRGNB nonduplicate isolates of ESBL positive
The reference BMD test was performed using a polystyrene microplate according to CLSI recommendations. The minimal inhibitory concentration of colistin sulfate salt (Sigma-AldrichC4461, USA) and polymyxyn B sulfate (Koçak, Turkey) were determined by BMD using cation-adjusted Mueller Hinton Broth (CAMHB) according to CLSI guidelines [6]. Each well of the microplate was filled with 100 μl CAMHB containing 0.5 to 512 μg/mL concentrations of colistin or polymyxin B and stocked at − 80°C until the working day (1–4 days). Stock strains were inoculated to sheep blood agar and incubated overnight at 37°C. Strains were prepared according to 0.5 McFarland turbidity on the working day, and they were adjusted to 106 CFU/mL by dilution. Then, 100 μl of bacteria were inoculated into the 1–11 wells, which were previously inoculated with 100 μl CAMHB containing 0.5 to 512 μg/mL concentrations of colistin or polymyxin B. The final concentration of antibiotics was 0.25–256 μg/mL. The twelfth well contained only bacteria and was used as positive control.
The MIC study for both antibiotics (colistin and polymyxin B) was performed at the same time using the same bacterial concentration. Since EUCAST does not have break points for the interpretation of polymyxin B, the interpretation of results for colistin and polymyxin B for
Colistin and polymyxin B evaluation criteria for Enterobacterales,
CLSI | Susceptible ( |
Intermediate( |
Resistant( |
||
---|---|---|---|---|---|
Colistin | ≤2 | ≥4 | |||
Polymyxin B | ≤2 | ≥4 | |||
EUCAST | Susceptible (mg/L) | Intermediate (mg/L) | Resistant (mg/L) | ECOFF (mg/L) | |
Colistin* | ≤2 | ≥4 | |||
Colistin** | ≤4 | ≥8 | |||
Polymyxin B* | 2 | ||||
Polymyxin B** | 4 |
The broth microdilution test was repeated when the MICs could not be read, the quality control results were not within the expected range, and the results were inconsistent (n = 63). The EA (MIC results within ± 1 doubling dilution) and CA (number of overlapping intermediate (I) and resistant (R) results between polymyxin B and colistin) were evaluated according to ISO criteria (EA and CA > 90%)[7].
Of the 268 strains, 109 (40.7%) were found to be colistin resistant and 91 (34.0%) polymyxin B resistant. According to the evaluation made with reference to colistin MIC results, EA, CA ratios for all isolates were found to be 81.3%, 85.1%, respectively.
The same MIC values were found for colistin and polymyxin B in 104 isolates (38.8%), and this rate was 31.1% (n=47) and 48.7% (n= 57) in
The essential agreement, categorical agreement rates of polymyxin B and resistance rates of colistin and polymyxin B in studied bacteria
Colistin | Polymyxin B | |||||
---|---|---|---|---|---|---|
Bacteria | EA n (%) | CA n (%) | I n (%) | R n (%) | I n (%) | R n (%) |
47 (79.7) | 39 (66.1) | 44 (74.6) | 15 (25.4) | 48 (81.4) | 11 (18.6) | |
75 (81.5) | 89 (96.7) | 30 (32.6) | 62 (67.4) | 33 (35.9) | 59 (64.1) | |
57 (90.5) | 54 (85.7) | 48 (76.2) | 15 (23.8) | 51 (81.0) | 12(19.0) | |
39 (72.2) | 46 (85.2) | 37 (68.5) | 17 (31.5) | 45 (83.3) | 9 (16.7) | |
Total | 218 (81.3) | 228 (85.1) | 159 (59.3) | 109 (40.7) | 177 (66.0) | 91 (34.0) |
Differences in MIC values between colistin and polymyxin B among tested isolates
Organisms (n) | Polymyxin B more potent by n (%) of strains | Same MIC | Colistin more potent by n (%) of strains | ||||
---|---|---|---|---|---|---|---|
3 dilution | 2 dilution | 1 dilution | n (%) | 1 dilution | 2 dilution | 3 dilution | |
0 | 4 (6.8) | 19 (32.2) | 17 (28.8) | 11 (18.6) | 7 (11.9) | 1 (1.7) | |
2 (2.2) | 13 (14.1) | 39 (42.4) | 30 (32.6) | 6 (6.5) | 2 (2.2) | 0 | |
1 (1.6) | 4 (6.3) | 15 (23.8) | 38 (60.3) | 4 (6.3) | 1 (1.6) | 0 | |
6 (11.1) | 9 (16.7) | 18 (33.3) | 19 (35.2) | 2 (3.7) | 0 | 0 | |
Total 268 | 9 (3.4) | 30 (11.2) | 91 (34.0) | 104 (38.8) | 23 (8.6) | 10 (3.7) | 1 (0.4) |
Categorical agreement rates changing according to susceptibility status for colistin-polymyxin B intermediate–resistant isolates
Strains | Colistin | Polymyxin B | % of Categorical agreement |
---|---|---|---|
44 | 36 | 81.8 | |
15 | 3 | 20 | |
30 | 30 | 100 | |
62 | 59 | 95.2 | |
74 | 66 | 89.2 | |
77 | 62 | 80.5 | |
48 | 45 | 93.8 | |
15 | 9 | 60 | |
37 | 37 | 100 | |
17 | 9 | 52.9 | |
85 | 82 | 96.5 | |
32 | 18 | 56.3 |
Colistin and polymyxin B resistance rates of
MIC values (μg/ml) for colistin and polymyxin B in
Grey font numbers indicate the number of strains with equivalent MICs for colistin and polymyxin B
Colistin (mg/L) | >=256 | 1K | ||||||||||
128 | 1K | 2K | 1K | |||||||||
64 | 4K | 9K | 5K | |||||||||
32 | 3K | 12K | 8K | 1K | ||||||||
16 | 4K | 4K | ||||||||||
8 | 1K+1E | 1K | 1K | |||||||||
4 | 1K+3E | 1K+8E | 1K+3E | 1K | ||||||||
2 | 2k | 2K | 5K+9E | 2K+7E | 2E | |||||||
1 | 1k | 2K+2E | 2K+4E | 1K+9E | 5E | |||||||
0.5 | 2k | 3K+2E | 1K | 2K+2E | 1E | |||||||
<=0.25 | 3k+1E | 2K | ||||||||||
0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | >=256 | ||
Polymyxin B (mg/L) |
MIC values (μg/ml) for colistin and polymyxin B in
Grey font numbers indicate the number of strains with equivalent MICs for colistin and polymyxin B.
Colistin (mg/L) | >=256 | 2A | ||||||||||
128 | 1A | |||||||||||
64 | 1P+1A | |||||||||||
32 | 1A | |||||||||||
16 | 1A | 1P+1A | ||||||||||
8 | 1P+2A | 1P | 2P | |||||||||
4 | 3A | 2P+2A | 2P+1A | 5P+1A | 1A | |||||||
2 | 1A | 1P+5A | 10P+8A | 16P+9A | 3P | |||||||
1 | 1A | 4A | 12P+4A | 1P+1A | ||||||||
0.5 | 1P+2A | 1P+2A | ||||||||||
0.25 | 2P | 1P | ||||||||||
0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | >=256 | ||
Polymyxin B (mg/L) |
In our study, the MIC value of polymyxin B was found to be lower than colistin in 39.0% (n = 23) of
MIC values and interpretation of colistin and polymyxin B against all strains studied are given in Table 7.
MIC values and interpretations of colistin and polymyxin B against
Bac. | Col. MIC | Interp. | Poly. MIC | Int. | Bac. | Col. MIC | Interp. | Poly. MIC | Interp. | Bac. | Col. MIC | Int. | Poly. MIC | Int. | Bac. | Col. MIC | Int. | Poly. MIC | Int. | Bac. | Col. MIC | Int. | Poly. MIC | Int. |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
E1 | 1 | I | 2 | I | K1 | 1 | I | 1 | I | K64 | 64 | R | 64 | R | P1 | 2 | I | 1 | I | A1 | 4 | R | 0.5 | I |
E2 | 1 | I | 4 | R | K2 | 1 | I | 1 | I | K65 | 64 | R | 32 | R | P2 | 4 | R | 1 | I | A2 | 4 | R | 2 | I |
E3 | 4 | R | 1 | I | K3 | 1 | I | 2 | I | K66 | 64 | R | 32 | R | P3 | 1 | I | 1 | I | A3 | 8 | R | 1 | I |
E4 | 2 | I | 1 | I | K4 | 1 | I | 0.5 | I | K67 | 64 | R | 32 | R | P4 | 1 | I | 1 | I | A4 | 256 | R | 128 | R |
E5 | 1 | I | 2 | I | K5 | 1 | I | 0.25 | I | K68 | 64 | R | 16 | R | P5 | 2 | I | 1 | I | A5 | 2 | I | 0.5 | I |
E6 | 2 | I | 2 | I | K6 | 1 | I | 0.5 | I | K69 | 64 | R | 32 | R | P6 | 4 | R | 2 | I | A6 | 4 | R | 4 | R |
E7 | 1 | I | 4 | R | K7 | 2 | I | 1 | I | K70 | 64 | R | 16 | R | P7 | 2 | I | 4 | R | A7 | 2 | I | 1 | I |
E8 | 1 | I | 2 | I | K8 | 2 | I | 1 | I | K71 | 64 | R | 32 | R | P8 | 1 | I | 2 | I | A8 | 0.5 | I | 0.5 | I |
E9 | 1 | I | 2 | I | K9 | 2 | I | 1 | I | K72 | 128 | R | 64 | R | P9 | 1 | I | 1 | I | A9 | 2 | I | 0.25 | I |
E10 | 4 | R | 2 | I | K10 | 2 | I | 0.5 | I | K73 | 128 | R | 64 | R | P10 | 1 | I | 1 | I | A10 | 2 | I | 2 | I |
E11 | 1 | I | 1 | I | K11 | 2 | I | 1 | I | K74 | 128 | R | 32 | R | P11 | 1 | I | 1 | I | A11 | 2 | I | 1 | I |
E12 | 1 | I | 2 | I | K12 | 2 | I | 0.25 | I | K75 | 128 | R | 128 | R | P12 | 2 | I | 1 | I | A12 | 16 | R | 4 | R |
E13 | 2 | I | 2 | I | K13 | 2 | I | 0.25 | I | K76 | 32 | R | 16 | R | P13 | 2 | I | 1 | I | A13 | 2 | I | 2 | I |
E14 | 1 | I | 2 | I | K14 | 2 | I | 0.5 | I | K77 | 0.25 | I | 0.5 | I | P14 | 1 | I | 1 | I | A14 | 0.5 | I | 0.5 | I |
E15 | 0.5 | I | 2 | I | K15 | 2 | I | 1 | I | K78 | 0.25 | I | 0.5 | I | P15 | 4 | R | 1 | I | A15 | 2 | I | 1 | I |
E16 | 0.5 | I | 2 | I | K16 | 2 | I | 2 | I | K79 | 0.25 | I | 0.25 | I | P16 | 2 | I | 1 | I | A16 | 2 | I | 1 | I |
E17 | 1 | I | 1 | I | K17 | 2 | I | 2 | I | K80 | 0.25 | I | 0.25 | I | P17 | 16 | R | 16 | R | A17 | 1 | I | 0.5 | I |
E18 | 1 | I | 0.5 | I | K18 | 4 | R | 8 | R | K81 | 0.25 | I | 0.25 | I | P18 | 2 | I | 1 | I | A18 | 2 | I | 2 | I |
E19 | 2 | I | 1 | I | K19 | 4 | R | 1 | I | K82 | 0.5 | I | 0.25 | I | P19 | 2 | I | 2 | I | A19 | 1 | I | 2 | I |
E20 | 4 | R | 2 | I | K20 | 4 | R | 4 | R | K83 | 0.5 | I | 0.5 | I | P20 | 1 | I | 1 | I | A20 | 2 | I | 2 | I |
E21 | 2 | I | 2 | I | K21 | 4 | R | 2 | I | K84 | 0.5 | I | 0.5 | I | P21 | 1 | I | 1 | I | A21 | 0.5 | I | 0.25 | I |
E22 | 2 | I | 1 | I | K22 | 8 | R | 2 | I | K85 | 0.5 | I | 1 | I | P22 | 1 | I | 1 | I | A22 | 2 | I | 2 | I |
E23 | 1 | I | 2 | I | K23 | 8 | R | 8 | R | K86 | 0.5 | I | 0.5 | I | P23 | 1 | I | 1 | I | A23 | 2 | I | 1 | I |
E24 | 1 | I | 4 | R | K24 | 8 | R | 4 | R | K87 | 0.5 | I | 0.25 | I | P24 | 8 | R | 2 | I | A24 | 1 | I | 1 | I |
E25 | 4 | R | 2 | I | K25 | 16 | R | 8 | R | K88 | 0.5 | I | 2 | I | P25 | 8 | R | 4 | R | A25 | 1 | I | 1 | I |
E26 | 2 | I | 4 | R | K26 | 16 | R | 16 | R | K89 | 0.5 | I | 2 | I | P26 | 8 | R | 4 | R | A26 | 2 | I | 2 | I |
E27 | 1 | I | 4 | R | K27 | 16 | R | 16 | R | K90 | 32 | R | 32 | R | P27 | 2 | I | 0.5 | I | A27 | 4 | R | 1 | I |
E28 | 2 | I | 1 | I | K28 | 16 | R | 16 | R | K91 | 256 | R | 128 | R | P28 | 2 | I | 2 | I | A28 | 256 | R | 128 | R |
E29 | 8 | R | 2 | I | K29 | 16 | R | 8 | R | K92 | 16 | R | 8 | R | P29 | 8 | R | 1 | I | A29 | 16 | R | 16 | R |
E30 | 4 | R | 2 | I | K30 | 16 | R | 16 | R | P30 | 2 | I | 1 | I | A30 | 2 | I | 1 | I | |||||
E31 | 1 | I | 4 | R | K31 | 16 | R | 8 | R | P31 | 2 | I | 1 | I | A31 | 1 | I | 1 | I | |||||
E32 | 1 | I | 2 | I | K32 | 32 | R | 8 | R | P32 | 1 | I | 1 | I | A32 | 128 | R | 64 | R | |||||
E33 | 2 | I | 2 | I | K33 | 32 | R | 16 | R | P33 | 64 | R | 64 | R | A33 | 1 | I | 0.5 | I | |||||
E34 | 0.5 | I | 4 | R | K34 | 32 | R | 16 | R | P34 | 1 | I | 1 | I | A34 | 32 | R | 32 | R | |||||
E35 | 4 | R | 4 | R | K35 | 32 | R | 64 | R | P35 | 0.25 | I | 1 | I | A35 | 2 | I | 1 | I | |||||
E36 | 1 | I | 1 | I | K36 | 32 | R | 32 | R | P36 | 2 | I | 1 | I | A36 | 64 | R | 64 | R | |||||
E37 | 4 | R | 2 | I | K37 | 32 | R | 32 | R | P37 | 2 | I | 2 | I | A37 | 1 | I | 1 | I | |||||
E38 | 2 | I | 1 | I | K38 | 32 | R | 16 | R | P38 | 2 | I | 1 | I | A38 | 0.5 | I | 0.25 | I | |||||
E39 | 1 | I | 2 | I | K39 | 32 | R | 32 | R | P39 | 0.5 | I | 0.5 | I | A39 | 1 | I | 0.5 | I | |||||
E40 | 4 | R | 2 | I | K40 | 32 | R | 16 | R | P40 | 2 | I | 2 | I | A40 | 1 | I | 0.25 | I | |||||
E41 | 4 | R | 2 | I | K41 | 32 | R | 32 | R | P41 | 0.25 | I | 0.25 | I | A41 | 4 | R | 8 | R | |||||
E42 | 2 | I | 2 | I | K42 | 32 | R | 8 | R | P42 | 0.25 | I | 0.25 | I | A42 | 2 | I | 0.5 | I | |||||
E43 | 4 | R | 4 | R | K43 | 32 | R | 16 | R | P43 | 0.5 | I | 0.25 | I | A43 | 2 | I | 0.5 | I | |||||
E44 | 2 | I | 2 | I | K44 | 32 | R | 8 | R | P44 | 4 | R | 2 | I | A44 | 4 | R | 1 | I | |||||
E45 | 4 | R | 4 | R | K45 | 32 | R | 16 | R | P45 | 4 | R | 4 | R | A45 | 4 | R | 0.5 | I | |||||
E46 | 2 | I | 4 | R | K46 | 32 | R | 32 | R | P46 | 2 | I | 4 | R | A46 | 2 | I | 0.5 | I | |||||
E47 | 1 | I | 0.5 | I | K47 | 32 | R | 16 | R | P47 | 2 | I | 2 | I | A47 | 4 | R | 0.5 | S | |||||
E48 | 2 | I | 1 | I | K48 | 32 | R | 16 | R | P48 | 2 | I | 2 | I | A48 | 2 | I | 0.5 | I | |||||
E49 | 1 | I | 1 | I | K49 | 32 | R | 16 | R | P49 | 2 | I | 2 | I | A49 | 8 | R | 1 | I | |||||
E50 | 4 | R | 2 | I | K50 | 32 | R | 16 | R | P50 | 2 | I | 2 | I | A50 | 2 | I | 2 | I | |||||
E51 | 0.25 | I | 0.25 | I | K51 | 32 | R | 32 | R | P51 | 2 | I | 2 | I | A51 | 2 | I | 2 | I | |||||
E52 | 0.5 | I | 0.5 | I | K52 | 32 | R | 32 | R | P52 | 4 | R | 4 | R | A52 | 1 | I | 0.5 | I | |||||
E53 | 2 | I | 2 | I | K53 | 32 | R | 16 | R | P53 | 4 | R | 4 | R | A53 | 2 | I | 2 | I | |||||
E54 | 4 | R | 1 | I | K54 | 64 | R | 32 | R | P54 | 2 | I | 2 | I | A54 | 2 | I | 1 | I | |||||
E55 | 0.5 | I | 0.5 | I | K55 | 64 | R | 32 | R | P55 | 4 | R | 4 | R | ||||||||||
E56 | 2 | I | 1 | I | K56 | 64 | R | 16 | R | P56 | 2 | I | 2 | I | ||||||||||
E57 | 2 | I | 1 | I | K57 | 64 | R | 32 | R | P57 | 2 | I | 2 | I | ||||||||||
E58 | 2 | I | 1 | I | K58 | 64 | R | 16 | R | P58 | 2 | I | 2 | I | ||||||||||
E59 | 4 | R | 1 | I | K59 | 64 | R | 64 | R | P59 | 2 | I | 2 | I | ||||||||||
K60 | 64 | R | 64 | R | P60 | 2 | I | 2 | I | |||||||||||||||
K61 | 64 | R | 64 | R | P61 | 2 | I | 2 | I | |||||||||||||||
K62 | 64 | R | 32 | R | P62 | 2 | I | 4 | R | |||||||||||||||
K63 | 64 | R | 64 | R | P63 | 4 | R | 4 | R |
The use of colistin and polymyxin B in the treatment of MDRGND infections, including carbapenem resistance, has gained importance again. The chemical structure of colistin and polymyxin B are very similar to each other (differing in one amino acid and the fatty acyl moiety). Despite the different forms in clinical use, it is recommended to use sulfate salt for both drugs in susceptibility tests [8].
In our country, the interpretation of antibiotic susceptibility tests is based on EUCAST standards. However, since there is no breakpoint value for polymyxin B in EUCAST, MIC values recommended by CLSI were used in our study. Questions that cause disagreement among our guides are whether
When we compared the MIC values of both antibiotics, it was found that 109 (40.7%) strains were resistant to colistin, while polymyxin B resistance was 91 (34.0%). It is noteworthy that the resistance rates of colistin were higher in all bacterial groups than polymyxin B.
Gales et al. [9] found that polymyxin B and colistin were resistant at rates of 0.1% and 0.2% in
In the study conducted by Chew et al. [10] 67.1% of 76 carbapenem resistant
In all bacterial groups (
In our study, although colistin and polymyxin B were found to have the same efficacy in
Similar to our findings, Doymaz et al. [12] found polymyxin B to be more effective in their study, with 89 isolates.
When colistin and polymyxin B susceptibility were analyzed according to bacterial species, equal MIC values of both antibiotics were seen, with the highest in
When all bacteria were evaluated in our study, 228 (85.1%) of 268 isolates did not meet the ISO criteria, although they were categorically compatible. When the results were evaluated separately on the basis of bacteria, 89 (96.7%) of 92
The study of Süzük et al. [13] found the categorical agreement to be acceptable (98.4%, 99.5%, respectively) and higher than our study in their comparison study with 505
In a review by Humphries [14], it was reported that susceptibility test results for colistin and polymyxin B were compatible in some studies, and it was emphasized that the resistance rate to polymyxins was low in the strains used. These results support our interpretation that a comparison should be made with a close number of susceptible and resistant isolates.
In our study, essential agreement (90.5%) was found to be acceptable only for
In our study, with a relatively limited number of samples, we found that colistin and polymyxin B are not compatible with each other for clinically important Gram-negative bacteria.
Colistin and polymyxin B resistance rates were different in the same isolates.
Polymyxin B is more potent than colistin and has lower MIC values.
Categorical agreement rates differ in resistant or intermediate strains of bacteria.
Although categorical agreement was higher in intermediate isolates than in resistant strains, the rate of CA was below acceptable limits in other bacterial groups, except for
We concluded that the colistin and polymyxin B susceptibility results cannot be used interchangeably.